Project summary Systematic functional dissection of neuronal transcriptome diversity Cell type-specific alternative splicing (AS) enormously amplifies the neuronal transcriptome diversity. Proper regulation of such molecular complexity and its establishment during development is critical for the maturation of nerve cells and maintenance of their homeostasis. Multiple RNA-binding proteins (RBPs) have been identified to control neuron-specific splicing. We pioneered the development of an ?RBP-centric? strategy to reconstruct precisely the splicing regulatory networks of specific classes of neuronal RBPs using an integrative analysis framework that combines multiple modalities of experimental and computational data. These efforts generated prioritized lists of developmentally regulated exons that will be studied in details to improve our understanding of the functional importance of AS at various stages of neuronal differentiation. However, a major roadblock for the field is our current inability to efficiently interrogate the function of a vast number of splice variants. To fill in this gap, we propose to develop an ?exon-centric? strategy using an exon- specific genetic screen to dissect directly and systematically the functional role of specific splice variants during neural development. For a pilot study, our focus is to identify alternative exons that regulate axon morphogenesis in an in vitro model system of spinal motor neurons derived from mouse embryonic stem (mES) cells. In Aim 1, we will establish a large-scale genome-editing platform to delete individual alternative exons in mES cells through lentivirus-based, CRISPR/Cas9-mediated genome engineering. We designed a cloning strategy that will allow us to build a CRISPR library with a large pool of paired guide RNAs (gRNAs) targeting individual alternative exons to trigger specific exon deletion. Parameters for optimizing the complexity of the library, viral delivery, and efficiency of genome editing will be established. In Aim 2, we will perform a pilot screen of ~100 prioritized neuronal alternative exons and identify those important for axon outgrowth. To perform this screening based on analysis of neuronal morphology in the absence of a reliable reporter, we propose a strategy to derive clonal mutant mES cell populations from transduced cell pools in a high-throughput format. These clonal lines carrying individual mutations will be subject to paralleled neuronal differentiation, high-throughput imaging and phenotypic analysis. This strategy will allow sensitive detection of mutants showing fine morphological defects in axon growth, which will be genotyped and further validated. Our approach will therefore combine advantages of being both scalable and flexible. This study will establish a very effective method to extend our knowledge of gene function to the level of individual splice variants. This strategy can be readily adapted to study the molecular programs underlying neural differentiation, migration, and function in normal and pathological contexts.